Purpose To elucidate the role of cyclin-dependent kinase inhibitor 3 (CDKN3) in the differentiation of chicken preadipocyte, laying a theoretical foundation for elucidating the molecular mechanism of fat deposition.
Methods The fluorescent expression vector pECFP-CDKN3 was constructed, and the subcellular localization of CDKN3 protein was observed after transfection to the immortalized chicken preadipocyte cell line (ICP1). Sodium oleate was used to induce the differentiation of ICP1, and qRT-PCR detected the expression level of CDKN3 mRNA during differentiation. pCMV-HA-CDKN3 and pCMV-HA were transfected into ICP1, and differentiation was induced after 24 hours of transfection, adipocyte differentiation was detected by oil red O staining, and the mRNA expression levels of fat differentiation-related genes after overexpression of CDKN3 were detected by qRT-PCR.
Results The cells transfected with pECFP-CDKN3 mainly had cyan fluorescence signal in the nucleus, suggesting that chicken CDKN3 was localized in the nucleus. In the process of inducing the lipid differentiation of ICP1, the expression level of CDKN3 mRNA gradually increased. The results of oil red O staining showed that the aggregation of lipid droplets in overexpressing CDKN3 was reduced, and the expression levels of C/EBPα, PPARγ, FABP4 and FAS related to fat differentiation showed a downward trend, among which FAS decreased significantly (P<0.05) and PPARγ decreased extremely significantly (P<0.01).
Conclusion CDKN3 is a nuclear protein that can inhibit the differentiation of chicken preadipocyte.
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